ABSTRACT Circular RNAs (circRNAs) are an emerging class of RNA molecules with potential for prolonged expression due to their inaccessibility to exonucleases. Recent studies dissecting circRNA biogenesis have found inverted repeat sequences such as ALU repeats in humans, flanking a large number of exons that are subject to circularization and have shown these cis-elements are essential for their circularization. CircRNA formation also requires both components of the standard pre-mRNA splicing machinery and canonical splicing signals, such as the polypyrimidine tract, and branch point. Although they have been largely demonstrated to have regulatory potential, recent studies have demonstrated that they can be translated. In the mammalian brain, circRNAs appear to be highly abundant and dynamically regulated by development and plasticity. In particular, they appear to be enriched at the synapses and during neuronal differentiation and development. Despite these exciting advances, no systems for efficient biogenesis of synthetic circRNAs in animal models have been developed to date. Host factors involved in regulating the formation of circRNAs in a spatio-temporal manner are also not well understood. Specific aims for the current proposal are focused on engineering circRNA expression systems, studying factors affecting circRNA biogenesis in the brain and the efficiency as well as potential toxicity of circRNA biogenesis in a mouse model of RNA-induced neurotoxicity. Overall, the current proposal will help develop new strategies for efficient biogenesis of circRNAs in animal models, which are likely to help understand factors affecting circRNA biogenesis in the mammalian brain as well as usher in a new class of nucleic acid therapeutics based on circular RNAs.